Process for determining the status of an organism by peptide measurement

ABSTRACT

A method for detecting the condition of an organism through the measurement of peptides from a sample of said organism containing high- and low-molecular weight peptides, as an indication of the condition of said organism, wherein
         low-molecular weight peptides are directly detected and characterized; and   related to a reference.

This application is the National Stage of PCT/EP97/04396, filed Aug. 13,1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for detecting the condition ofan organism through the measurement of peptides from a sample of saidorganism.

2. The Prior Art

Various analytical methods are employed for detecting the condition ofan organism. Thus, for example, in the diagnostics of higher organisms,when pathological results are obtained, attempts are made to fathom thecauses of the pathological change on the basis of the symptoms in orderto develop a causal therapy. Further, efforts are being made to developa reference of an average “healthy” organism by sequencing the genomesof organisms and establishing “wild type genomes” in order to be able todiscover individual deviations which could indicate possible pathogenicdevelopments by performing corresponding gene analyses. A drawback ofthe first methodological approach is that diagnostics free fromhypotheses (bias-free) cannot be performed since the diagnostics thereinare already based on assumptions. A drawback of the second method isthat it will not be possible for a long time to diagnose the importantor even all diseases attributed to genetic dysfunctions. Anotherdrawback of the latter method may also be that a mutation on a gene doesnot necessarily result in expression of the related phenotype.

Thus, it would be desirable to provide a universally employablediagnostic method by which it is possible to avoid the drawbacksmentioned and, in particular, to perform diagnostics free fromhypotheses. In addition, the diagnostic method should be universallyemployable, not be restricted to higher developed systems, but also beemployable for detecting the condition of lower organisms. In addition,it should be easy to establish and capable of being carried out with perse known techniques.

SUMMARY OF THE INVENTION

Thus, it has been the object of the present invention to provide such amethod.

Surprisingly, the object of the invention is achieved in a simple mannerby a method with the features according to the invention.

The method according to the invention for detecting the condition of anorganism starts by taking a sample from the organism to be examined.This sample may also be the complete organism. The sample must containlow-molecular weight peptides, but there is no interference fromhigh-molecular weight peptides or proteins which are also contained inthe sample in addition to low-molecular weight peptides. According tothe invention, the low-molecular weight peptides are directly detectedand characterized and serve as indicators of the condition of theorganism. It is possible to detect single peptides directly by ameasuring technique, to detect several peptides by a measuringtechnique, or even all the low-molecular weight peptides present in thesample which can be detected by a measuring technique. Unlikeconventional analytical or diagnostic methods, such as gelelectrophoresis or two-dimensional electrophoresis and, for example,clinical diagnostic methods, the method according to the invention doesnot examine the high-molecular weight structures, such as proteins. Asopposed to per se known diagnostic methods, such as radioimmunoassay orother competitive assays for the measurement of peptide hormones and thelike, the low-molecular weight peptides are directly detected accordingto the invention by some measuring technique rather than indirectly asin the methods mentioned. The distribution of low-molecular weightpeptides in a representative cross-section of defined controls is usedas a reference.

In the method according to the invention, the sample to be examined maybe derived from tissue or fluid samples from the organism the conditionof which is to be detected, or it may be the organism itself or partsthereof. When lower organisms are examined, the organism itself ispreferably used as the sample. Such lower organisms include, inparticular, single-celled organisms, such as procaryotic systems orsimple eucaryotic systems, such as yeasts or other microorganisms.

According to the invention, the low-molecular peptides employed formeasurement shall preferably have a molecular weight of not more than30,000 Dalton. The lower limit is not actually critical, but dipeptidesrepresent the lower limit of low-molecular weight peptides to bedetected according to the invention. Particularly preferred aremolecular weights of the low-molecular weight peptides of from 100 to10,000 Dalton.

If required, for example, due to a changed measuring arrangement, it maybe advantageous to remove high-molecular weight peptides or proteins andother biopolymers which might interfere with the measurement from thesample. This is not required, in particular, in cases where thehigher-molecular weight peptide compounds are not covered by themeasuring method to be employed according to the invention.

Preferably, according to the invention, mass spectroscopy is employedfor detecting the low-molecular weight peptides. Particularly preferredis the so-called MALDI method (matrix assisted laser desorptionionization mass spectroscopy). If mass spectroscopy is employed as amethod, it is recommendable to employ the data obtainable by said massspectroscopy for characterizing the low-molecular weight peptides, suchas their molecular weights. It is also possible, under particularcircumstances, to analyze other parameters, such as the charge of thepeptides, or the characteristic retention times on chromatographiccolumns, or a fragment pattern of the low-molecular weight peptides, orcombinations of the mass and charge of the low-molecular weightpeptides.

Depending on the additional questions connected with the detection ofthe condition of the organism, it may be advantageous to divide thesample into several fractions and to analyze the samples under differentaspects or with different measuring arrangements, and thus to detect acondition of the organism.

The organisms include, in particular, procaryotes, eucaryotes,multicellular organisms, cells from tissue cultures, cells from animalsand humans. Thus, it becomes possible according to the invention toexamine the condition of genetically engineered or transformed and/orconditioned organisms. This may be advantageous, in particular, forchecking transformed systems in order to recognize any unexpected orundesirable properties which might have been developed by transformedorganisms, for instance, by forming peptides indicative of undesirableor unexpected properties, such as toxic properties.

In particular, any deliberately or unintentionally performedmanipulation (conditioning) of an organism may influence its condition,whether during the administration of medicaments, gene therapy,infections, in the working place from contact with chemical substances,in test animals, especially transgenic animals and knock-out mutants.Especially in the case of such methods, an intra- and interindividualcomparison, for example, through the chronological taking of samplesfrom an organism prior to and in the course of one of the abovementioned measures, or a comparison with untreated control organism maybe used to check whether the predicted and desired changes in conditionhave actually occurred, and whether, in addition or instead,unpredicted, undesirable or desirable, changes have occurred which aredetected by the method according to the invention without the need torecur to hypotheses.

Therefore, the method according to the invention is also useful, forexample, for accompanying clinical studies, toxicological examinationsin the testing of medicaments of all kinds, for analyzing/detectingdecomposition products, for the identification of gene products.

In veterinary and human medicine, the method according to the inventiongains its outstanding importance by the fact that it enables thedetection of the condition of the respective organism without the needto recur to hypotheses. Thus, rather than performing a confirmationassay based upon a preconceived opinion, a real overall picture of thecondition of the organism examined can be created. The method accordingto the invention, which may be designated as a differential peptidedisplay, is based on the fact that a particular peptide pattern ispresent in a healthy organism which is therefore capable of serving as areference standard. Now, if the peptide condition of an individual isrecorded and compared to that of the reference, deviations can bedetected which provide a first indication of a possibly pathogeniccondition. By detecting the deviations established by comparison withsimilar pathogenic conditions from corresponding samples of a diseased,it is then possible to identify the respective disease directly from theanalysis by a mere comparison of the deviations in the peptide patternof the sample of said individual, and correspondence of the deviationwith an assigned clinical picture.

According to the invention, one may proceed as follows, in particular.Ultrafiltrates from body fluids and tissue extracts may first be usedfor preparing a reference sample. Recovery of the filtrate peptides andtheir separation into fractions is performed, for example, by collectinglow-molecular weight peptide fractions. The characterization of thepeptide fractions may be effected, for example, by their retentionbehavior and molecular weight, which can be determined by chromatographyor mass spectroscopy. For example, if an ultrafiltrate from patientssuffering from a known disease is used and compared with the previouslyestablished spectrum of healthy reference subjects, the deviatingpattern enables an assignment of the specific disease to the conditionof the respective peptide mixture. Thus, this method may also beemployed in a per se conventional manner, for example, by immediatelyinterrogating the appropriate peptide pattern indicative of pathogenicchanges. In some cases, this may even be one peptide characteristic ofthe respective disease. For example, if a sample is analyzed from apatient for whom a particular clinical picture can be recognized and ahypothesis for the cause of such disease exists, this specific peptidemay also be interrogated in the analysis according to the invention, andif the result is positive, appropriate therapeutic schemes may beestablished. Thus, it is altogether possible to first take a sample fromthe patient, to record a condition by the method according to theinvention, and then, if the presence of a deviation indicative ofpathogenic conditions is established, either to perform a controlmeasurement by per se known confirmation assays recurring to the usualclinical assays, or to perform such control measurement by specificallyscreening for the indicator of the pathogenic condition.

Peptides may be recovered by methods known to those skilled in the art,such as ultrafiltration of the respective starting material. When doingso, filters are used having a molecular exclusion size within the rangeclaimed according to the invention, i.e., between that of a dipeptideand a maximum of 30,000 Dalton. By appropriately selecting therespective membranes, it is also possible to obtain fractions ofparticular molecular weights. Preferably, from 0.2 ml to 50 l offiltrate is obtained from the filtration, which is adjusted, forexample, to a pH value of from 2 to 4 by acidification with dilutedhydrochlorid acid immediately after the end of the filtration. Theamounts mentioned especially serve to examine pooled samples, fordeveloping reference samples from healthy subjects, or for determiningdisease-specific peptide markers for establishing a peptide data base.

The peptides present in the filtrate after ultrafiltration are recoveredby adsorption to chromatographic materials, especially cationexchangers, such as Fractogel, anion exchangers-Fractogel TMAE, andreversed phase (RP) materials, followed by elution with linear gradientsor step gradients. For further purification, other chromatographicseparations, especially through reversed phase materials, may optionallybe effected.

The measurement of the peptide fractions is preferably performed bymass-spectrometrical analysis, especially with MALDI MS (matrix assistedlaser desorption ionization mass spectrometry) or ESI MS (electrosprayionization MS). These are methods which can be used for the analysis ofpeptides. This preferably involves the on-line coupling of Microborereversed phase separation and mass spectrometry (LC-MS coupling). Fromthe data obtained, a multidimensional table is established based onretention behavior, molecular weight and signal intensity as thepreferred guiding parameters. However, other quantities which can bedetermined with the mentioned methods may also be recorded.

The data about patients with a known basic disease obtained from theabove mentioned steps are compared to the similarly obtained data from ahealthy reference population. Both qualitative changes (e.g., theoccurrence of new peptides or the lacking of peptides) and quantitativechanges (the increased or decreased occurrence of individual peptides)are detected. If required, the targets defined by the comparativeanalysis may further be purified and identified by methods of peptidechemistry known to those skilled in the art. The sequence informationobtained can then be compared with protein and nucleic acid data basesand subsequently with data from the literature. The relevance of therepresented peptides with respect to the examined disease is checked byfunctional studies and by screenings with appropriate groups ofpatients.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1 Use of BodyFluids: Blood Filtrate (Hemofiltrate, HF)

1. Recovery of HF

HF is recovered by arterio-venous or veno-venous hemofiltrationperformed by techniques known to those skilled in the art with selectedpatients or subjects. The recovery of HF is effected in the same way, inprinciple, as performed as a matter of a routine in patients withchronical renal disease. Through an arterial drain and venous feed(arterio-venous hemofiltration) or venous drain and venous feed(veno-venous hemofiltration), the patient's blood is passed with the aidof a hemofiltration device (e.g., Hemoprozessor, Sartorius, Göttingen;AK 10 HFM, Gambro, Hechingen) through a hemofilter (e.g., Hemoflow F 60or Hemoflow HF 80 S, Fresenius, Bad Homburg; Hemoflow FH 77 H andHemoflow HF 88 H, Gambro) which has a molecular exclusion size of up to30 kDa. The filtrate volume withdrawn from the patient is substituted byan electrolyte solution (e.g., SH 01, SH 05, SH 22, SH 29, Schiwa,Glandorf).

According to the present method, a diagnostic hemofiltration isperformed with the aim to obtain from 1 to 30 1 of HF from a patient inthe course of one hemofiltration. For avoiding proteolysis, thehemofiltrate is immediately adjusted to a pH value between 2 and 4 withdiluted acid (e.g., 1 M HCl), and cooled to 4° C.

2. Recovery of the HF Peptides and Separation Into Fractions

2.1 Peptide Extraction with Stepwise Elution

10 l of hemofiltrate is diluted with deionized water to provide aconductivity of 6 mS/cm, and its pH value is adjusted to 2.7 withhydrochlorid acid. The HF is then applied to a chromatographic column.After binding of the HF peptides, the bound peptides are eluted with apH step elution using 7 buffers with increasing pH values.

Chromatographic conditions:

-   -   flow for application: 100 ml/min    -   flow for elution: 30 ml/min    -   detection: 214, 280 nm    -   column: Vantage (Amicon, Witten), 6 cm diameter×7 cm filling        height    -   column material: Fraktogel TSK SP 650 M (Merck, Darmstadt)    -   equipment: BioCAD 250, Perseptive Biosystems,        Wiesbaden-Nordenstadt

buffer pH value buffer substances molarity elution buffer 1 3.6 citricacid. 0.1 elution buffer 2 4.5 acetic acid 0.1 elution buffer 3 5.0malic acid 0.1 elution buffer 4 5.6 succinic acid 0.1 elution buffer 56.6 sodium dihydrogenphosphate 0.1 elution buffer 6 7.4 disodiumhydrogenphosphate 0.1 elution buffer 7 9.0 ammonium carbonate 0.1

Eluates 1-7 are separately collected.

2.2 Second Chromatographic Separation

Eluates 1-7 are separately subjected to chromatography through areversed phase column.

Chromatographic conditions:

-   -   flow for application: 10 ml/min    -   flow for elution: 4 ml/min    -   detection: 214 nm    -   column: HPLC steel column, 1 cm diameter, 12.5 filling height    -   column material: Source RPC 15 μm (Pharmacia, Freiburg)    -   equipment: BioCAD, Perseptive Biosystems, Wiesbaden-Nordenstadt

The eluate is collected in 4 ml fractions.

3. Mapping of the Peptide Fractions.

3.1

Aliquots of the fractions obtained in 2.2 are applied to a Microborereversed phase column and eluted in a gradient. Detection is effectedwith a UV detector and on-line with an electrospray mass spectrometer.

Chromatographic conditions:

-   -   flow for application: 20 μl/min    -   flow for elution: 20 μl/min    -   detection: 220 nm    -   column: C18 AQS, 3 μm, 120 A, 1 mm diameter, 10 cm length (YMC,        Schermbeck)    -   equipment: ABI 140 B Dual Solvent Delivery System    -   buffer A: 0.06% trifluoroacetic acid in water    -   buffer B: 80% acetonitrile in A    -   gradient: 0% B to 100% B in 90 min

On-line mass spectrometry:

API III with electrospray interface (Perkin-Elmer, Weiterstadt) positiveion mode

-   -   measuring range:m/z from 300 to 2390    -   scan time: 7 s    -   scan window: 0.25 m/z

Data acquisition is performed with MacSpec or MultiView Soft-ware(Perkin-Elmer).

3.2 MALDI MS Measurement of the Individual Fractions

Aliquots of the fractions obtained in 2.2 are measured with differentmatrix substances, e.g., with the addition of L-(−)-fucose, in MALDI MS.

From the raw data, a multidimensional table is established consideringthe scan number, signal intensity and, after calculation, of the massesfrom the multiple-charged ions of a scan.

4. Comparative Analysis

4.1 Identification of Novel or Lacking Peptides or Those SignificantlyDeviating in Quantity

By comparing the data sets obtained under 3.3, which may also bereferred to as peptide maps, qualitative and/or quantitative differencesare established. Considering controls and samples, individual data setsor sets of data sets are used for comparison.

4.2 Peptide-chemical Characterization of the Identified Targets

From the raw material obtained (e.g., large preparations ofhemofiltrate), the identified targets are purified in such amounts asallow identification, using the different chromatographic separationtechniques known to those skilled in the art which are generallyemployed for separating peptide mixtures (reversed phase, ion-exchange,size exclusion, hydrophobic interaction, etc.). After eachchromatographic separation of a fraction, the targets are againidentified in the fractions by ESI MS, MALDI MS or LC MS. This procedureis repeated, with variation of the chromatographic parameters, until apure product of the desired specification, i.e., retention time andmolecular weight, has been obtained. This is followed by thedetermination of a partial or complete amino acid sequence or a fragmentpattern. Subsequently, a data base comparison is performed with theknown data bases (Swiss-Prot and EMBL-Peptid- undNucleinsäure-Datenbank), with the object to identify the partial orcomplete sequence or a fragment pattern. If no data base entry exists,the primary structure is clarified.

EXAMPLE 2 Use of Body Fluids: Ascitic Fluid

1. Recovery of Ascitic Fluid

Ascitic fluid is formed as an extravascular exsudate in various diseases(malignant tumors, liver disorders etc.). According to the presentmethod, between 10 ml and 10 1 of ascitic fluid is obtained by punctionand then immediately adjusted to a pH value of between 2.0 and .4.0with: diluted acid (e.g., 1 M HCl) in order to avoid proteolysis, andcooled to 4° C. After ultrafiltration over a cellulose triacetatemembrane with an exclusion size of 30 kDa (Sartocon mini-apparatus,Sartorius), the filtrate is further used as a source of peptides.

2. Recovery of the Ascitic Fluid Peptides and Separation into Fractions

2.1 Peptide Extraction with Gradient Elution

5 of ascitic fluid filtrate is adjusted to pH 2.0 and separated througha preparative reversed phase column.

Chromatographic conditions:

-   -   flow for application: 40 ml/min    -   flow for elution: 40 ml/min    -   detection: 214 nm, 280 nm    -   column: Waters cartridge system, 4.7 cm diameter, 30 cm filling        height    -   column material: Vydac RP-C18, 15-20 μm    -   equipment: BioCAD, Perseptive Biosystems, Wiesbaden-Nordenstadt    -   buffer A: 0.1% trifluoroacetic acid in water    -   buffer B: 80% acetonitrile in A    -   gradient: 0% B to 100% B in 3000 ml

The eluate is collected in 50 ml fractions.

The further course of the characterization corresponds to that inExample 1.

EXAMPLE 3 Use of Body Fluids: Urine

1. Recovery of Urine

Urine is directly recovered as catheter urine or spontaneous urine frompatients in amounts of from 0.5 to 50 1 and immediately adjusted to a pHvalue of between 2.0 and 4.0 with diluted acid (e.g., 1 M HCl) in orderto avoid proteolysis, and cooled to 4° C. After ultrafiltration over acellulose triacetate membrane with an exclusion size of 30 kDa (Sartoconmini-apparatus, Sartorius), the filtrate is further used as a source ofpeptides.

2. Recovery of the Urine Peptides and Separation into Fractions

2.1 Peptide Extraction with Stepwise Elution

10 l of urine filtrate is diluted with water to provide a conductivityof 6 mS/cm, and its pH value is adjusted to 2.7 with HCl. The urinefiltrate is then applied to a chromatographic column. After binding ofthe peptides, the bound peptides are eluted with a saline gradient.

Chromatographic conditions:

-   -   flow for application: 100 ml/min    -   flow for elution: 30 ml/min    -   detection: 214 nm    -   column: Vantage (Amicon, Witten), 6 cm diameter×7 cm filling        height    -   column material: Merck Fraktogel TSK SP 650 M    -   equipment: BioCAD 250, Perseptive Biosystems,        Wiesbaden-Nordenstadt    -   buffer A: 50 mM NaH₂PO₄, pH 3.0    -   buffer B: 1.5 M NaCl in A    -   gradient: 0% B to 100% B in 2000 ml

The eluate is collected in 10 pools of 200 ml each.

2.2 Second Chromatographic Separation

The fractions are separately subjected to chromatography through areversed phase column.

Chromatographic conditions:

-   -   flow for application: 10 ml/min    -   flow for elution: 4 ml/min    -   detection: 214 nm    -   column: HPLC steel column, 1 cm diameter, 12.5 cm filling height    -   column material: Pharmacia Source RPC 15 μm    -   equipment: BioCAD, Perseptive Biosystems, Wiesbaden-Nordenstadt    -   buffer A: 0.1% trifluoroacetic acid in water    -   buffer B: 80% acetonitrile in A    -   gradient: 0% B to 100% B in 200 ml

The eluate is collected in 4 ml fractions.

The further course of the characterization corresponds to that inExample 1.

1. A method for detecting a pathogenic conditions in an organismcomprising the steps of: measuring all low-molecular weight peptideshaving a molecular weight of not more than 30,000 Daltons detectable byMALDI mass spectrometry present in a sample of body fluid taken fromsaid organism by directly detecting said low-molecular weight peptidesby MALDI mass spectrometry; to provide a distribution of low-molecularweight peptides; and relating said low-molecular weight peptides to areference comprising a distribution of low-molecular weight peptides ina representative cross-section of defined controls of said organism toproduce a differential peptide display; wherein said body fluid sampleis selected from the group consisting of a hemofiltrate, an asciticfluid, and urine, wherein said organism is an animal or a human, andwherein said differential peptide display is indicative of a pathogeniccondition.
 2. The method according to claim 1, wherein said detectedlow-molecular weight peptides have a molecular weight of from 100 to10,000 Daltons.
 3. The method according to claim 1, whereinhigh-molecular weight peptides having a molecular weight of greater than30,000 Daltons are also present in said sample and said high-molecularweight peptides are either separated off prior to measurement of saidlow-molecular weight peptides, or left unconsidered, in terms ofmeasurement or evaluation, in the recording of the sample.
 4. The methodaccording to claim 1, wherein said sample is divided into differentfractions prior to said measurement of the low-molecular weightpeptides, and the fractions are measured under different detectionconditions.
 5. A method of detecting the physiological condition of anorganism without reference to a preconceived diagnosis comprising thesteps of: (a) directly measuring the distribution of all detectablelow-molecular weight peptides in a sample from a test organism; and (b)comparing the distribution measured in step (a) to a distribution oflow-molecular weight peptides from a sample of a reference organism toprovide a differential peptide display illustrating the differences inlow-molecular weight peptide distribution between the test organism andthe reference organism; wherein the low-molecular weight peptidesmeasured have a molecular weight of not more than 30,000 Daltons; saiddistributions of low-molecular weight peptides are directly measured bymass spectrometry; and wherein the differential peptide display providesfor detection of the physiological condition of the test organismwithout reference to a preconceived diagnostic hypothesis regarding thecondition of the test organism.
 6. The method of claim 5 wherein thedistributions of low-molecular weight peptides are measured by MALDImass spectrometry or electrospray ionization mass spectrometry.
 7. Themethod of claim 5 wherein the samples are separated into fractions byliquid chromatography prior to measuring the low-molecular weightpeptide distributions.
 8. The method of claim 5 wherein thelow-molecular weight peptides measured have a molecular weight of from100 to 10,000 Daltons.
 9. The method of claim 5 wherein the testorganism and reference organism are both humans or are both the samespecies of animal.
 10. The method of claim 9 wherein the referenceorganism is a normal, healthy organism, and wherein the differentialpeptide display indicates whether the test organism has a physiologicalcondition that differs from the physiological condition of the healthyreference organism.
 11. The method of claim 10 wherein the differentialpeptide display is compared to differential peptide displays fromorganisms with known pathological conditions in order to diagnosewhether the test organism has a known pathological condition.
 12. Themethod of claim 5 wherein the samples are ultrafiltrates of a bodilyfluid from the organism selected from the group consisting of ahemofiltrate, a urine ultrafiltrate, and an ascitic fluid ultrafiltrate.13. The method of claim 12 wherein the ultrafiltrate is obtained byfiltering the bodily fluid through a size exclusion membrane having anexclusion size of 30,000 Daltons.
 14. The method of claim 5 wherein thesample of the reference organism is from the same individual testorganism and the differential peptide display indicates a physiologicalchange in the test organism over a period of time.
 15. The method ofclaim 5 wherein the test organism is a genetically engineered organismand the reference organism is a genetic control organism; and whereinthe differential peptide display indicates whether the geneticallyengineered organism exhibits an unpredicted, undesirable or desirablephysiological change relative to the genetic control reference organism.16. The method of claim 5 wherein high-molecular weight peptides arealso present in said sample and said high-molecular weight peptides areeither separated off prior to measurement of said low-molecular weightpeptides, or left unconsidered, in terms of measurement or evaluation,in the recording of the sample.
 17. A method of detecting a pathologicalcondition in a human or animal without reference to a preconceiveddiagnosis comprising the steps of: (a) directly measuring thedistribution of all detectable low-molecular weight peptides in a samplefrom a test organism; (b) comparing the distribution measured in step(a) to a distribution of low-molecular weight peptides from a sample ofa reference organism to provide a differential peptide displayillustrating the differences in low-molecular weight peptidedistribution between the test organism and the reference organism; and(c) comparing the differential peptide display in step (b) with adifferential peptide display from an organism having a knownpathological condition; wherein each of the test organism, the referenceorganism, and the organism having a known pathological condition is ahuman or each is an animal; the low-molecular weight peptides measuredhave a molecular weight of not more than 30,000 Daltons; saiddistributions of low-molecular weight peptides are directly measured bymass spectrometry; and wherein the comparison in step (c) provides fordiagnosis of a pathological condition in the test organism withoutreference to a preconceived diagnostic hypothesis regarding thecondition of the test organism.
 18. The method of claim 17, wherein thelow-molecular weight peptides measured have a molecular weight of from100 to 10,000 Daltons.
 19. The method of claim 17 wherein the samplesare separated into fractions by liquid chromatography prior to measuringthe low-molecular weight peptide distributions.
 20. The method of claim17 wherein the samples are ultrafiltrates of a bodily fluid from theorganism selected from the group consisting of a hemofiltrate, a urineultrafiltrate, and an ascitic fluid ultrafiltrate.
 21. The method ofclaim 20 wherein the ultrafiltrate is obtained by filtering the bodilyfluid through a size exclusion membrane having an exclusion size of30,000 Daltons.
 22. The method of claim 17 wherein the distributions oflow molecular weight peptides are measured by MALDI or electrosprayionization mass spectrometry.
 23. The method of claim 17 whereinhigh-molecular weight peptides are also present in said sample and saidhigh-molecular weight peptides are either separated off prior tomeasurement of said low-molecular weight peptides, or left unconsidered,in terms of measurement or evalution, in the recording of the sample.